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Departments of
*
Microbiology,
Pathology, and
Medicine, University of Alabama, Birmingham, AL 35294; and
Institute for Molecular Medicine for the Prevention of Human Diseases, University of Texas-Houston Health Science, Houston, TX 77030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To overcome the limitations of exogenously administered complement inhibitors and Ab-dependent disease models, we chose to study myelin oligodendrocyte glycoprotein (MOG)-induced EAE in mice deficient for C3 (C3-/-) or factor B (FB-/-). MOG-induced EAE has been shown to be Ab-independent in the C57BL/6 mouse strain when induced using the encephalitogenic peptide 35-55 (21, 22). Here, we show that C3-/- and FB-/- mice are largely protected from myelin damage and develop less severe clinical signs of the disease. This effect is accompanied by an unusual distribution of inflammatory cells, characterized by a minimal intraparenchymal infiltration of the CNS. In particular, both C3-/- and FB-/- mice induced for EAE present with decreased numbers of infiltrating macrophages, T cells, and ICAM-1+ cells in the CNS compared with complement-sufficient mice. These results show unequivocally that complement contributes to the pathogenesis of MOG-induced EAE in mice and supports the notion that complement inhibitors may be useful in the treatment of MS.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The C3-/- and FB-/- mice used have been described previously (23, 24) and were originally generated in the 129SVJ/C57BL/6 (H2-Db/H2-Db) background. The C3-/- mice produce no serum C3 and consequently lack the ability to generate the anaphylatoxin C3a and the opsonin C3b, and have no serum complement lytic activity (23). The FB-/- mice produce no serum FB and thus cannot form the alternative pathway C3 convertase, rendering them devoid of alternative pathway lytic activity (24). These mice also have reduced classical pathway activity (24). The C3-/- and FB-/- mice were backcrossed to C57BL/6 for at least five generations before the induction of EAE, and their complement-sufficient littermates served as controls. It should be noted that the 129SVJ strain is EAE resistant (25); thus, the clinical presentation of EAE that we observed in this study is a trait of the susceptible C57BL/6 strain. Additional experiments (n = 22 mice) performed for another study confirmed a 100% incidence of EAE in C3-/- and FB-/- mice bearing the C57BL/6-backcrossed background (data not shown). Screening of complement-deficient mice was performed by PCR amplification of the targeted DNA sequence from samples of DNA extracted from tail biopsies (23, 24) and by assessment of serum C3 and FB levels by ELISA (24, 26).
EAE induction and evaluation
In all EAE experiments, C3-/- and FB-/- mice were compared with and immunized at the same time as their wild-type littermates expressing one or two copies of the normal gene. All mice used in this study were females between 8 and 12 wk of age at the time of immunization. Mice were immunized with the MOG peptide 35-55 as previously described (27). Briefly, MOG peptide was synthesized by standard 9-fluorenyl-methoxycarbonyl chemistry and was shown to be >95% pure as determined by reversed phase-HPLC (Research Genetics, Huntsville, AL). Mice were then injected s.c. on days 0 and 7 with 150 µg of peptide emulsified in CFA. In addition, on days 0 and 2 postimmunization (p.i.), mice were given pertussis toxin (500 ng) i.p. Clinical signs of EAE were assessed daily using a standard scale of 0–6 as follows: 0, no clinical signs; 1, loss of tail tone; 2, flaccid tail; 3, incomplete paralysis of one or two hind legs; 4, complete hind limb paralysis; 5, moribund; and 6, death. Animals showing clinical signs of grade 5 for >2 consecutive days were sacrificed and assigned a score of 6. For each animal immunized for EAE, a mean cumulative disease index (CDI) was calculated from the sum of the daily clinical scores observed between day 1 p.i. and day 21 p.i. The average maximum clinical score was calculated for each phenotype group from the sums of the highest clinical score for each mouse.
Histological assessment
At day 21 p.i. or between days 30 and 34 p.i., mice were sacrificed by CO2 inhalation, and spinal cords were removed and either fixed with 4% paraformaldehyde and 2% glutaraldehyde or snap frozen and kept at -80°C until examination. Five to seven animals were randomly chosen in each experimental group (C3- and FB-sufficient controls, FB-/-, and C3-/-), and their spinal cords were assessed for inflammation and demyelination. First, 8-µm frozen sections were stained with hematoxylin and eosin for initial assessment of inflammation. In parallel, the extent of demyelination was evaluated by toluidine blue staining on 1-µm sections of lumbo-thoracic spinal cords embedded in Epon. The presence or absence of demyelination was further confirmed by Luxol fast blue-cresyl violet stains for myelin, and the demyelinated nature of identified lesions was verified by demonstrating axonal integrity within the lesions using modified Bielschowsky stains of adjacent sections. For each technique, four to six sections were evaluated blindly for demyelination and inflammation by three examiners (S.N., S.R.B., and S.L.C.). Demyelination was scored from 0 (normal white matter) to +++ (extensive demyelination), and inflammation was evaluated in different anatomical compartments (meninges, parenchyma, and vessels). Inflammation was scored using the following scale: for meninges and parenchyma: 0, no infiltrating cells; +, few infiltrating cells; ++, numerous infiltrating cells; and +++, widespread infiltration; for vessels: 0, no cuffed vessel; +, one or two cuffed vessels per section; ++, three to five cuffed vessels per section; and +++, more than five cuffed vessels per section.
Immunohistochemistry
C3-/- mice (n = 3) and FB-/- mice (n = 3) and their complement protein-sufficient littermates (n = 3 and 2, respectively) were immunized for EAE and sacrificed on day 30 p.i. to collect lumbo-thoracic spinal cords for immunohistochemical analysis. Immunohistochemistry was performed on 10-µm-thick frozen transversal sections using the Vectastain avidin-biotin complex kit (Vector Laboratories, Burlingame, CA). Acetone-fixed sections were incubated for 60 min with mouse anti-CD11b mAb (BD PharMingen, San Diego, CA), rat anti-mouse CD3 monoclonal Ab (Serotec, Kidlington, U.K.), or hamster anti-mouse ICAM-1 monoclonal Ab (BD PharMingen, San Diego, CA). Biotin-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, Baltimore, MD) or a biotin-conjugated "universal" Ab (Vector Laboratories) was then applied. Sections were then rinsed in PBS, incubated in a solution of 1% hydrogen peroxide for 15 min, and washed. Finally, the sections were treated with avidin-peroxidase for 50 min at room temperature (Vectastain avidin-biotin complex kit; Vector Laboratories) followed by addition of 0.04% diaminobenzamadine (Sigma, St. Louis, MO) in PBS with 0.01% H2O2 for 10 min. Semiquantitative analysis of CD11b, CD3, and ICAM-1 was performed by two examiners, and cells were counted as described: three to six sections per animal were examined at low magnification (x20), and the three lesions showing the strongest staining were analyzed at higher magnification (x100) to count labeled cells. Data obtained from C3-and FB-sufficient controls (n = 3 and 2, respectively) were pooled and are referred to as results from EAE control mice. These results were compared with those obtained from C3-/- (n = 3) and FB-/- mice (n = 3).
Electron microscopy
For one animal in each experimental group, ultrathin sections of lumbo-thoracic spinal cords were stained with toluidine blue and white matter tracts were examined by electron microscopy.
Statistical analysis
Results were analyzed for statistical significance using Student’s t test with p < 0.05 considered to be significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All immunized controls (n = 18) presented with
clinical signs of EAE starting on average at day 11 p.i. and with
the clinical peak of disease occurring at days 17–21 (Fig. 1
). In contrast, the incidence of disease
did not reach 100% in complement-deficient animals as one of six of
the C3-/- and two of eight of the
FB-/- mice did not show any clinical
signs of EAE during the 30-day survey period. Furthermore, the clinical
severity of EAE was attenuated in both C3-/-
and FB-/- mice compared with their wild-type
littermates (Fig. 1). FB-/- mice had a lower
maximum clinical score (2.7 vs 3.9; p = 0.03) and a
lower CDI (15.6 vs 27.4; p = 0.03) compared with those
of controls. Similarly the maximum clinical score was significantly
lower in C3-/- mice compared with controls
(2.75 vs 4.7; p = 0.04) (Table I). The mean clinical scores (Fig. 1)
were significantly reduced in both C3-/- (2.1
vs 3.45; p = 0.01) and FB-/-
mice (1.96 vs 2.83; p = 0.04).
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With the exception of one C3-/- mouse, all
mice immunized for EAE demonstrated CNS inflammation as assessed by
hematoxylin and eosin and toluidine blue staining (Table I
). On
examination of the different CNS compartments (meninges, Virchow-Robin
spaces, vessels, perivascular space, and parenchyma), we found cellular
infiltration throughout the CNS in C3- and FB-sufficient controls
(Figs. 2, A and C,
3, A and C, and
Table I). In contrast, although there was significant meningeal
infiltration and perivascular cuffing in both
C3-/- and FB-/- mice,
we observed virtually no infiltration in the parenchyma (Figs. 2B and 3B; Table I). We also evaluated the extent
of demyelination in control and complement-deficient mice by toluidine
blue staining of lumbo-thoracic spinal cord sections obtained from
three animals per experimental group. In addition, Luxol fast
blue-cresyl violet stains were performed on spinal cord sections of two
FB-/- and two FB control animals immunized for
EAE and sacrificed at day 34 p.i. In control groups, extensive
areas of subpial, perivascular, and parenchymal demyelination were
observed associated with parenchymal infiltrating cells (Figs. 2, C and E, 3, C and E, and
Table I). In contrast, in both C3-/- and
FB-/- mice immunized for EAE, only a few areas
of limited subpial demyelination were noticed in all sections examined
(Figs. 2D and 3D; Table I). Interestingly, in
these animals, the loss of myelin sheaths in areas surrounding severe
meningeal and/or perivascular infiltration appeared very mild or
negligible (Figs. 2F and 3F). These data were
supported by electron microscopy analysis of spinal cord sections
obtained from EAE control animals which showed extensive myelin
destruction along with astrogliosis and parenchymal infiltration by
multivacuolar macrophage cells containing lipid droplets (Fig. 4, A and B). In
contrast, C3-/- and
FB-/- mice, sacrificed at the same time point
after induction of EAE, had only mild or no myelin damage (Fig. 4C).
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To determine whether there were differences in the composition of
the cellular infiltrate among control, C3-/-,
and FB-/- mice, we performed
immunohistochemical analysis. In control EAE mice, numerous
CD3+ T cells were detected in the meninges,
perivascular space, and parenchyma of the spinal cord (Fig. 5A). Compared with control
mice, C3-/- mice presented with remarkably
fewer CD3+-infiltrating cells (Fig. 5B
and Table II). Comparable results were
obtained using FB-/- mice (data not shown).
Infiltrating cells expressing the macrophage marker CD11b were also
detected in the spinal cords of control EAE mice and most of these
cells displayed a round morphology (Fig. 5C). It has been
demonstrated that during EAE, CD11b+ round cells
comprise macrophages and ameboid microglia with high phagocytic
activity whereas CD11b+ process-bearing cells are
putative microglial cells with low phagocytic activity
(28). Interestingly, although there was a decrease in
CD11b+ cells in C3-/- EAE
mice compared with control EAE mice, the number of
CD11b+ round cells compared with
CD11b+ process-bearing cells was significantly
reduced (Fig. 5D and Table II). Similar results were
obtained using FB-/- mice (data not shown). We
also observed a reduction in ICAM-1+ staining in
C3-/- or FB-/- mice on
both infiltrating cells and putative endothelial cells (Table II).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to altered cellular trafficking, we observed a remarkable
reduction in demyelination in FB-/- and
C3-/- mice compared with controls.
Demyelination involves macrophage- and microglia-mediated removal of
myelin sheaths and engulfment of myelin debris, a process considered to
be largely complement dependent (9, 10, 44, 45).
Furthermore, it is established that the complement receptor type 3
(CR3, CD18/CD11b) plays a pivotal role in myelin phagocytosis during
EAE, with ligand binding resulting in increased phagocytosis as well as
TNF-
and NO release by macrophages and microglia (45, 46). Obviously the limited complement activation potential of
FB-/- and C3-/- mice
would partially or completely inhibit the generation of the
complement-derived opsonin C3b. As we observed little to no
demyelination in both types of complement-deficient mice, our data
demonstrate an essential role for C3 and factor B in this process. The
role C3a plays in glial cell chemoattraction is unclear, but may be
less important than C5a as microglia are not chemoattracted by C3a in
vitro (42).
It is noteworthy that despite the absence of key complement components,
FB-/- and C3-/- mice
developed EAE (albeit in an attenuated form). C5a, and perhaps C3a, may
still contribute to disease development and progression in the
complement-deficient mice, despite their reduced levels, as it is
possible that proteases other than the C3 and C5 convertases might
generate small amounts of these potent inflammatory mediators. For
example, it has been shown that trypsin, -thrombin, plasmin,
elastase, and kallikrein can generate C5a or C5a-like fragments with
functional activity (47, 48, 49). Whether C3a or C3a-like
molecules can also be generated by similar mechanisms is not
established. Thus, the possibility that complement anaphylatoxins,
especially C5a, might be released through bypass mechanisms in
C3-/- and FB-/- mice
during EAE cannot be excluded.
We have recently shown that CNS-targeted expression of a soluble complement inhibitor, sCrry, prevents or delays the onset of MOG-induced EAE (50). sCrry transgenic mice with a C57BL/6 background have delayed disease onset, but then develop EAE with similar severity to that observed in their nontransgenic littermates. In contrast, sCrry-transgenic mice with a SJL background (a strain which develops a milder form of EAE) had no clinical signs of disease. We report here that for MOG-induced EAE in FB-/- and C3-/- C57BL/6 mice, disease onset was identical to that of complement-sufficient controls, but disease severity was significantly reduced. The combined data from both the sCrry-transgenic mice and the complement-deficient mice demonstrate that inhibiting complement-mediated functions at multiple levels in the complement activation pathways provides significant protection from MOG-induced EAE. Moreover, our data indicate that the alternative pathway might be the predominate pathway involved in complement-mediated inflammation and demyelination in MOG-induced EAE. Preliminary studies from our laboratory show that treatment with the chimeric recombinant complement inhibitor Crry-Ig (51) also provides significant protection from MOG-induced EAE (data not shown). Although these data indicate that inhibition of complement activation provides protection in the effector phase of disease, it is likely that complement-mediated functions are important in the inductive phase of disease as well. The increased expression of the C5aR on CNS endothelium and on infiltrating cells before clinical signs of disease (7), and the marked attenuation of EAE severity in C5aR-/- mice (43) support this notion. Taken together, these data strengthen the concept that intervention in the complement system provides a potentially beneficial therapeutic avenue for the treatment of MS and other CNS inflammatory diseases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Scott R. Barnum, Department of Microbiology, University of Alabama, Birmingham, 701 19th Street South, LHR/141, Birmingham, AL 35294.
3 Abbreviations used in this paper: MS, multiple sclerosis; EAE, experimental allergic encephalomyelitis; CVF, cobra venom factor; MOG, myelin oligodendrocyte glycoprotein; FB factor B; p.i., postimmunization; CDI, cumulative disease index.
Received for publication June 13, 2000. Accepted for publication August 17, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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suppresses CR3-mediated myelin removal by macrophages. J. Neuroimmunol. 38:9.[Medline]
and nitric oxide. J. Neuroimmunol. 70:145.[Medline]
. J. Immunol. 162:2321. and nitric oxide. J. Neuroimmunol. 70:145.
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